Fishing for pearls more efficiently with a new NMR method

NMR is the most venerable approach for finding fragments, and ligand-detected NMR is still among the more popular methods. But the amount of protein required for a full fragment library screen can be a limitation, particularly for more challenging targets. A new paper in Angew. Chem. Int. Ed. by Alvar Gossert and collaborators at ETH Zürich, Bruker, and Karlsruhe Institute of Technology provides a new, less protein-intensive approach.

 

I’ll preface the next paragraph by admitting that not only am I no spectroscopist, I don’t even play one on TV. So, spectroscopy-savvy readers, please feel free to provide more details in the comments, especially if I get something wrong. For fellow non-spectroscopists, the takeaway is that clever NMR tricks increase sensitivity.

 

PEARLScreen, short for Perfect Echo for Advanced Relaxation-based Ligand Screen, is related to the classic Carr-Purcell-Meiboom-Gill (CPMG, or T_1ρ) method, which we wrote about most recently here. As in that older method, PEARLScreen relies on the decrease in signal intensity of a ligand that binds to a protein. This is due to slower tumbling of the bound ligand, resulting in faster relaxation of excited protons (see here). Lengthening the time between excitation and measurement should in theory boost contrast between bound and free ligands, but various technical challenges impede this in practice. PEARLScreen overcomes these challenges using “a perfect echo pulse train with water suppression by excitation sculpting.” In addition to lengthening the relaxation delay, PEARLScreen also allows exchange broadening to occur between the ligand and protein, further increasing sensitivity.

 

The researchers simulated multiple conditions to optimize various parameters, and then experimentally tested PEARLScreen on four different proteins with three types of NMR instruments, starting with a standard high-end 600 MHz.

 

The first protein-ligand pair was trypsin binding to a known benzamidine fragment. This interaction was detectable using a standard T_1ρ experiment with 200 µM ligand and 20 µM protein. Using PEARLScreen, the researchers could reduce the protein concentration to 1 µM while maintaining similar signal to noise .

 

Next, they screened 94 fragments in pools of 8 against three different proteins: PPAT, Abl, and FKBP. In all cases PEARLScreen was more sensitive than T_1ρ, allowing screening at 2.5 µM rather than 20 µM protein. PEARLScreen was also more sensitive than the two other most common ligand-detected NMR methods, STD and WaterLOGSY.

 

We wrote recently about benchtop NMR, and the researchers found that PEARLScreen was also more sensitive than a T_1ρ experiment on an 80 MHz instrument, though the difference was not as dramatic as on the 600 MHz machine. On the other hand, on a 1.2 GHz instrument PEARLScreen was so sensitive that the researchers could screen mixtures of 16 fragments with just 1 µM protein.

 

This is a neat paper, which confidently concludes that “due to the superior sensitivity of the PEARLScreen compared to all established screening experiments at standard fields, we expect it to become the standard experiment for ¹H-detected ligand screening.” We look forward to hearing how it performs for others.